Journal: Cell reports
Article Title: PhosY-secretome profiling combined with kinase-substrate interaction screening defines active c-Src-driven extracellular signaling
doi: 10.1016/j.celrep.2023.112539
Figure Lengend Snippet: (A) Domain structure of human c-Src protein (UniprotKB: P12931) with key regulatory sites illustrated. From the N to the C terminus, domains include the N-term SRC homology SH4 with the U (brown), SH3 (blue), SH2 (orange), KD (SH1; green), and C-term R (red) domains. Domain range is indicated with residue numbers shown below each domain (above for SH3). Regions targeted by the mAbs tested in this study are indicated (black brackets). One asterisk (*) indicates mAbs that inhibit TIMP2 tyrosine phosphorylation and TIMP2 interaction with MMP-2. Two asterisks (**) indicate the mAb6 did not block TIMP2 phosphorylation or its interaction with MMP2 in experiments performed in this figure. (B) Schematic representation of sample preparation and processing for evaluating anti-c-Src Abs in cells.
(C and D) SYF+c-Src in (C) or HT1080 in (D) cells were seeded for 18 h, followed by serum starvation for 24 h. Cells were pretreated for 1 h with indicated anti-c-Src Ab or IgG as a control, followed by incubation with recombinant TIMP2-His for 2 h. Cell extracts and CMs were collected and analyzed by western blot and pull-down experiments. (C) SYF+c-Src cells were pretreated with anti-c-Src mAb1 (clone 32G6, biotinylated), mAb2 (clone 327), or IgG isotype control (clone DA1E, biotinylated) or were left untreated. Following incubation with recombinant TIMP2-His 6 (rTIMP2-His 6 ), TIMP2-His 6 was pulled-down from CM using Ni-NTA resin. TIMP2 phosphorylation was evaluated using anti-pan-phosY antibody (4G10). Copull-down of MMP2 and e-Src was determined by immunoblot. GAPDH (cell extracts) was used as loading control (Lys C). (D) HT1080 cells were pretreated with anti-c-Src mAb6 (clone 327537), mAb5 (clone 32G6), or IgG isotype control (clone 20102) or were left untreated. Following incubation with rTIMP2-His 6 , TIMP2-His 6 was pulled down from CM. TIMP2 phosphorylation was evaluated using anti-pan-phosY antibody (4G10). Copull-down of MMP2 was determined by immunoblot. (E) Schematic representation c-Src SH4/U and SH3 domains. The critical region used as immunogen for generating the polyclonal Ab (aa 84–110) in this study is highlighted in pink. (F) HT1080 cells were pretreated with anti-c-Src pAb (aa 84–110) or IgG isotype control (clone ERP25A) or were left untreated. Following incubation with rTIMP2-His 6 , TIMP2-His 6 was pulled down from CM. TIMP2 phosphorylation was evaluated using anti-pan-phosY antibody (4G10). Copull-down of MMP2 was determined by immunoblot. (G) Schematic representation of sample preparation for (H). Prostate cancer cells (LNCaP, DU-145, PC-3) were seeded in a 96-well plate for 18 h, followed by the addition (treatment) of the anti-c-Src Ab or IgG isotype control (clone ERP25A). After 72 h, cell proliferation was measured using a WST assay. (H) Proliferation assay on prostate cancer cells treated with the indicated concentration of antibody or IgG was measured by WST assay. The graph shows the percentage of growth normalized to the untreated sample. Data are presented as mean ± SEM derived from two technical replicates. Data presented are a representative result of 3 independent experiments. Paired two-tailed t test was used to assess statistical significance between IgG treatment and anti-c-Src treatment at 50 μg/mL for each cell line (*p < 0.05, **p < 0.01). SE, short exposure; LE, long exposure. See also .
Article Snippet: Rabbit anti-Src mAb1 (32G6, biotinylated) , Cell Signaling Technology , Cat#8077; RRID:AB_10860048.
Techniques: Residue, Phospho-proteomics, Blocking Assay, Sample Prep, Control, Incubation, Recombinant, Western Blot, WST Assay, Proliferation Assay, Concentration Assay, Derivative Assay, Two Tailed Test
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